Requirement for functional DNA polymerase eta in genome-wide repair of UV-induced DNA damage during S phase

The autosomal recessive disorder Xeroderma pigmentosum-variant (XPV) is characterized (i) at the cellular level by dramatic hypermutability and defective recovery of DNA synthesis following UV exposure, and (ii) clinically by abnormal sunlight sensitivity and remarkable predisposition to skin cancer. These phenotypes are clearly attributable to germline mutations in POLH, encoding DNA polymerase eta (polη) normally required for accurate translesion DNA synthesis (TLS) past UV-induced cyclobutane pyrimidine dimers. Here we demonstrate that patient-derived XPV-skin fibroblasts exposed to 15 J/m² of UV also exhibit (in addition to abnormal TLS) a significant defect in global-genomic nucleotide excision repair (GG-NER) exclusively during S phase. This cell cycle-specific GG-NER defect can be complemented by ectopic expression of wild-type polη, but not of polη variants deficient in either nuclear relocalization or PCNA interaction. We highlight a previous study from our laboratory demonstrating that UV-exposed, ATR-deficient Seckel syndrome fibroblasts, like XPV fibroblasts, manifest strong attenuation of GG-NER uniquely in S phase populations. We now present further evidence suggesting that deficient S phase repair can be rescued in both XPV- and Seckel syndrome-cells if the formation of blocked replication forks post-UV is either prevented or substantially reduced, i.e., following, respectively, pharmacological inhibition of DNA synthesis prior to UV irradiation, or exposure to a relatively low UV dose (5 J/m²). Our findings in cultured cells permit speculation that abrogation of GG-NER during S phase might partially contribute (in a synergistic manner with defective, atypically error-prone TLS) to the extreme state of UV-hypermutability leading to accelerated skin cancer development in XPV patients. Moreover, based on the overall data, we postulate that loss of either functional polη or -ATR engenders abnormal persistence of stalled replication forks at UV-adducted sites in DNA which, in turn, can actively and/or passively trigger GG-NER inhibition.     

Overproduction of DNA polymerase eta does not raise the spontaneous mutation rate in diploid human fibroblasts

Telomerase-immortalized lines of diploid xeroderma pigmentosum variant (XP-V) fibroblasts (XP115LO and XP4BE) were complemented for constitutive or regulated expression of wild-type human DNA polymerase eta (hpol eta). The ectopic gene was expressed from a retroviral LTR at a population average of 34- to 59-fold above the endogenous (mutated) mRNA and high levels of hpol eta were detected by immunoblotting. The POLH cDNA was also cloned downstream from an ecdysone-regulated promoter and transduced into the same recipient cells. Abundance of the wild-type mRNA increased approximately 10-fold by addition of ponasterone to the culture medium. Complemented cell lines acquired normal resistance to the cytotoxic effects of UVC, even in the presence of 1mM caffeine. They also tolerated higher levels of UVC-induced template lesions during nascent DNA elongation when compared to normal fibroblasts (NHF). UVC-induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus were measured in the XP115LO+XPV cell line overproducing hpol eta constitutively (E. Bassett, N.M. King, M.F. Bryant, S. Hector, L. Pendyala, S.G. Chaney, M. Cordeiro-Stone, The role of DNA polymerase eta in translesion synthesis past platinum-DNA adducts in human fibroblasts, Cancer Res. 64 (2004) 6469-6475). Induced mutation frequencies were significantly reduced, even below those observed in NHF; however, the average mutation frequency in untreated cultures was about three-fold higher than in the isogenic vector-control cell line. In this study, spontaneous HPRT mutation frequencies were measured at regular intervals, as isogenic fibroblasts either lacking or overproducing hpol eta were expanded for 100 population doublings. The mutation rates estimated from these results were not significantly increased in XP115LO cells expressing abnormal levels of hpol eta, relative to the cells lacking this specialized polymerase. These findings suggest that diploid human fibroblasts with normal DNA repair capacities and intact checkpoints are well protected against the potential mutagenic outcome of overproducing hpol eta, while still benefiting from accurate translesion synthesis of UV-induced pyrimidine dimers.     

Deficiency of the Caenorhabditis elegans DNA polymerase eta homologue increases sensitivity to UV radiation during germ-line development

Defects in the human XPV/POLH gene result in the variant form of the disease xeroderma pigmentosum (XP-V). The gene encodes DNA polymerase eta (Poleta), which catalyzes translesion synthesis (TLS) past UV-induced cyclobutane pyrimidine dimers (CPDs) and other lesions. To further understand the roles of Poleta in multicellular organisms, we analyzed phenotypes caused by suppression of Caenorhabditis elegans POLH (Ce-POLH) by RNA interference (RNAi). F1 and F2 progeny from worms treated by Ce-POLH-specific RNAi grew normally, but F1 eggs laid by worms treated by RNAi against Ce-POLD, which encodes Poldelta did not hatch. These results suggest that Poldelta but not Poleta is essential for C. elegans embryogenesis. Poleta-targeted embryos UV-irradiated after egg laying were only moderately sensitive. In contrast, Poleta-targeted embryos UV-irradiated prior to egg laying exhibited severe sensitivity, indicating that Poleta contributes significantly to damage tolerance in C. elegans in early embryogenesis but only modestly at later stages. As early embryogenesis is characterized by high levels of DNA replication, Poleta may confer UV resistance in C. elegans, perhaps by catalyzing TLS in early embryogenesis.     

Replication of damaged DNA: molecular defect in xeroderma pigmentosum variant cells.

Individuals with Xeroderma pigmentosum (XP) syndrome have a genetic predisposition to sunlight-induced skin cancer. Genetically different forms of XP have been identified by cell fusion. Cells of individuals expressing the classical form of XP (complementation groups A through G) are deficient in the nucleotide excision repair (NER) pathway. In contrast, the cells belonging to the variant class of XP (XPV) are NER-proficient and are only slightly more sensitive than normal cells to the killing action of UV light radiation. The XPV fibroblasts replicate damaged DNA generating abnormally short fragments either in vivo [A.R. Lehmann, The relationship between pyramidine dimers and replicating DNA in UV-irradiated human fibroblasts, Nucleic Acids Res. 7 (1979) 1901-1912; S.D. Park, J.E. Cleaver, Postreplication repair: question of its definition and possible alteration in Xeroderma pigmentosum cell strains, Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 3927-3931.] or in vitro [S.M. Cordeiro, L.S. Zaritskaya, L.K. Price, W.K. Kaufmann, Replication fork bypass of a pyramidine dimer blocking leading strand DNA synthesis, J. Biol. Chem. 272 (1997) 13945-13954; D.L. Svoboda, L.P. Briley, J.M. Vos, Defective bypass replication of a leading strand cyclobutane thymine dimer in Xeroderma pigmentosum variant cell extracts, Cancer Res. 58 (1998) 2445-2448; I. Ensch-Simon, P.M. Burgers, J.S. Taylor, Bypass of a site-specific cis-syn thymine dimer in an SV40 vector during in vitro replication by HeLa and XPV cell-free extracts, Biochemistry 37 (1998) 8218-8226.], suggesting that in XPV cells, replication has an increased probability of being blocked at a lesion. Furthermore, extracts from XPV cells were found to be defective in translesion synthesis [A. Cordonnier, A.R. Lehmann, R.P.P. Fuchs, Impaired translesion synthesis in Xeroderma pigmentosum variant extracts, Mol. Cell. Biol. 19 (1999) 2206-2211.]. Recently, Masutani et al. [C. Masutani, M. Araki, A. Yamada, R. Kusomoto, T. Nogimori, T. Maekawa, S. Iwai, F. Hanaoka, Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity, EMBO J. 18 (1999) 3491-3501.] have shown that the XPV defect can be corrected by a novel human DNA polymerase, homologue to the yeast DNA polymerase eta, which is able to replicate past cyclobutane pyrimidine dimers in DNA templates. This review focuses on our current understanding of translesion synthesis in mammalian cells whose defect, unexpectedly, is responsible for the hypermutability of XPV cells and for the XPV pathology.     

Low synthesis of retinoic acid due to impaired cytochrome P450 1a1 expression in mouse xeroderma pigmentosum fibroblasts

New tumor formation was suppressed by retinoic acid (RA) administration in xeroderma pigmentosum (XP) patients who have a defect in nuclear excision repair. However, the inhibition is not due to enhanced removal of UV-damaged DNA. These results prompted us to investigate whether or not RA metabolism is abnormal in XP fibroblasts and what the underlying mechanism is. Compared with wild type fibroblasts, low activities of RA synthesis were determined on HPLC in mouse fibroblasts lacking XP group A (XPA) gene and UV-induced XPA deficient cancer cells. Moreover, we observed an impaired expression of cytochrome P450 1a1 in XPA deficient fibroblasts by RT-PCR and a decreased expression of retinoic acid receptor gamma in XPA deficient cancer cells by Western blotting. Finally, pre-treatment of RA isoforms significantly protected the XPA deficient fibroblasts from UV-induced death. These results suggest that decreased structure activity of RA synthesis, resulting from impaired mRNA expression of cytochrome P450 1a1 may, at least together with UV irradiation, involve in skin carcinogenesis in XP patients.     

Polymerase eta and p53 jointly regulate cell survival,apoptosis and Mre11 recombination during S phase checkpoint arrest after UV irradiation

Xeroderma pigmentosum variant (XPV) cells lack the damage-specific polymerase eta and undergo a protracted arrest at the S phase checkpoint(s) following UV damage. The S phase checkpoints encompass several qualitatively different processes, and stimulate downstream events that are dependent on the functional state of p53. Primary fibroblasts with wild-type p53 arrest in S, and require a functional polymerase eta (pol eta) to carry out bypass replication, but do not recruit recombination factors for recovery. XPV cells with non-functional p53, as a result of transformation by SV40 or HPV16 (E6/E7), recruit the hMre11/hRad50/Nbs1 complex to arrested replication forks, coincident with PCNA, whereas normal transformed cells preferentially use the pol eta bypass replication pathway. The formation of hMre11 foci implies that arrested replication forks rapidly undergo a collapse involving double strand breakage and rejoining. Apoptosis occurs after UV only in cells transformed by SV40, and not in normal or XPV fibroblasts or HPV16 (E6/E7) transformed cells. Conversely, ultimate cell survival in XPV cells was much less in HPV16 (E6/E7) transformed cells than in SV40 transformed cells, indicating that apoptosis was not a reliable predictor of cell survival. Inhibition of p53 transactivation by pifithrin-alpha or inhibition of protein synthesis by cycloheximide did not induce hMre11 foci or apoptosis in UV damaged fibroblasts. Inhibition of kinase activity with wortmannin did not increase killing by UV, unlike the large increase seen with caffeine. Since HPV16 (E6/E7) transformed XPV cells were highly UV sensitive and not further sensitized by caffeine, it appears likely that caffeine sensitization proceeds through a p53 pathway. The S phase checkpoints are therefore, a complex set of different checkpoints that are coordinated by p53 with the capacity to differentially modulate cell survival, apoptosis, bypass replication and hMre11 recombination.     

Reduced efficiency and increased mutagenicity of translesion DNA synthesis across a TT cyclobutane pyrimidine dimer, but not a TT 6-4 photoproduct, in human cells lacking DNA polymerase eta

Xeroderma pigmentosum variant (XPV) patients carry germ-line mutations in DNA polymerase eta (poleta), a major translesion DNA synthesis (TLS) polymerase, and exhibit severe sunlight sensitivity and high predisposition to skin cancer. Using a quantitative TLS assay system based on gapped plasmids we analyzed TLS across a site-specific TT CPD (thymine-thymine cyclobutane pyrimidine dimer) or TT 6-4 PP (thymine-thymine 6-4 photoproduct) in three pairs of poleta-proficient and deficient human cells. TLS across the TT CPD lesion was reduced by 2.6-4.4-fold in cells lacking poleta, and exhibited a strong 6-17-fold increase in mutation frequency at the TT CPD. All targeted mutations (74%) in poleta-deficient cells were opposite the 3'T of the CPD, however, a significant fraction (23%) were semi-targeted to the nearest nucleotides flanking the CPD. Deletions and insertions were observed at a low frequency, which increased in the absence of poleta, consistent with the formation of double strand breaks due to defective TLS. TLS across TT 6-4 PP was about twofold lower than across CPD, and was marginally reduced in poleta-deficient cells. TLS across TT 6-4 PP was highly mutagenic (27-63%), with multiple mutations types, and no significant difference between cells with or without poleta. Approximately 50% of the mutations formed were semi-targeted, of which 84-93% were due to the insertion of an A opposite the template G 5' to the 6-4 PP. These results, which are consistent with the UV hyper-mutability of XPV cells, highlight the critical role of poleta in error-free TLS across CPD in human cells, and suggest a potential involvement, although minor, of poleta in TLS across 6-4 PP under some conditions.     

A non-catalytic role of DNA polymerase η in recruiting Rad18 and promoting PCNA monoubiquitination at stalled replication forks

Trans-lesion DNA synthesis (TLS) is a DNA damage-tolerance mechanism that uses low-fidelity DNA polymerases to replicate damaged DNA. The inherited cancer-propensity syndrome xeroderma pigmentosum variant (XPV) results from error-prone TLS of UV-damaged DNA. TLS is initiated when the Rad6/Rad18 complex monoubiquitinates proliferating cell nuclear antigen (PCNA), but the basis for recruitment of Rad18 to PCNA is not completely understood. Here, we show that Rad18 is targeted to PCNA by DNA polymerase eta (Polη), the XPV gene product that is mutated in XPV patients. The C-terminal domain of Polη binds to both Rad18 and PCNA and promotes PCNA monoubiquitination, a function unique to Polη among Y-family TLS polymerases and dissociable from its catalytic activity. Importantly, XPV cells expressing full-length catalytically-inactive Polη exhibit increased recruitment of other error-prone TLS polymerases (Polκ and Polι) after UV irradiation. These results define a novel non-catalytic role for Polη in promoting PCNA monoubiquitination and provide a new potential mechanism for mutagenesis and genome instability in XPV individuals.     

Retrovirus-mediated DNA repair gene transfer into xeroderma pigmentosum cells: Perspectives for a gene therapy

The rare hereditary disease xeroderma pigmentosum (XP) is clinically characterized by extreme sun sensitivity and an increased predisposition for developing skin cancer. Cultured cells from XP patients exhibit hypersensitivity to ultraviolet (UV) radiation due to the defect in nucleotide excision repair (NER), and other cellular abnormalities. Seven genes identified in the classical XP forms, XPA to XPG, are involved in the NER pathway. In view of developing a strategy of gene therapy for XP, we devised recombinant retrovirus-carrying DNA repair genes for transfer and stable expression of these genes in cells from XP patients. Results showed that these retroviruses are efficient tools for transducing XP fibroblasts and correcting repair-defective cellular phenotypes by recovering normal UV survival, unscheduled DNA synthesis, and RNA synthesis after UV irradiation, and also other cellular abnormalities resulting from NER defects. These results imply that the first step of cellular gene therapy might be accomplished successfully.     

Xeroderma pigmentosum variant and error-prone DNA polymerases

Replicative DNA synthesis is a faithful event which requires undamaged DNA and high fidelity DNA polymerases. If unrepaired damage remains in the template DNA during replication, specialised low fidelity DNA polymerases synthesises DNA past lesions (translesion synthesis, TLS). Current evidence suggests that the polymerase switch from replicative to translesion polymerases might be mediated by post-translational modifications involving ubiquitination processes. One of these TLS polymerases, polymerase eta carries out TLS past UV photoproducts and is deficient in the variant form of xeroderma pigmentosum (XP-V). The dramatic proneness to skin cancer of XP-V individuals highlights the importance of this DNA polymerase in cancer avoidance. The UV hypermutability of XP-V cells suggests that, in the absence of a functional poleta, UV-induced lesions are bypassed by inaccurate DNA polymerase(s) which remain to be identified.     

Diverse roles of RAD18 and Y-family DNA polymerases in tumorigenesis

Mutagenesis is a hallmark and enabling characteristic of cancer cells. The E3 ubiquitin ligase RAD18 and its downstream effectors, the ‘Y-family’ Trans-Lesion Synthesis (TLS) DNA polymerases, confer DNA damage tolerance at the expense of DNA replication fidelity. Thus, RAD18 and TLS polymerases are attractive candidate mediators of mutagenesis and carcinogenesis. The skin cancer-propensity disorder xeroderma pigmentosum-variant (XPV) is caused by defects in the Y-family DNA polymerase Pol eta (Polη). However it is unknown whether TLS dysfunction contributes more generally to other human cancers. Recent analyses of cancer genomes suggest that TLS polymerases generate many of the mutational signatures present in diverse cancers. Moreover biochemical studies suggest that the TLS pathway is often reprogrammed in cancer cells and that TLS facilitates tolerance of oncogene-induced DNA damage. Here we review recent evidence supporting widespread participation of RAD18 and the Y-family DNA polymerases in the different phases of multi-step carcinogenesis.     

Xeroderma pigmentosum complementation group E protein (XPE/DDB2): purification of various complexes of XPE and analyses of their damaged DNA binding and putative DNA repair properties

Xeroderma pigmentosum is characterized by increased sensitivity of the affected individuals to sunlight and light-induced skin cancers and, in some cases, to neurological abnormalities. The disease is caused by a mutation in genes XPA through XPG and the XP variant (XPV) gene. The proteins encoded by the XPA, -B, -C, -D, -F, and -G genes are required for nucleotide excision repair, and the XPV gene encodes DNA polymerase eta, which carries out translesion DNA synthesis. In contrast, the mechanism by which the XPE gene product prevents sunlight-induced cancers is not known. The gene (XPE/DDB2) encodes the small subunit of a heterodimeric DNA binding protein with high affinity to UV-damaged DNA (UV-damaged DNA binding protein [UV-DDB]). The DDB2 protein exists in at least four forms in the cell: monomeric DDB2, DDB1-DDB2 heterodimer (UV-DDB), and as a protein associated with both the Cullin 4A (CUL4A) complex and the COP9 signalosome. To better define the role of DDB2 in the cellular response to DNA damage, we purified all four forms of DDB2 and analyzed their DNA binding properties and their effects on mammalian nucleotide excision repair. We find that DDB2 has an intrinsic damaged DNA binding activity and that under our assay conditions neither DDB2 nor complexes that contain DDB2 (UV-DDB, CUL4A, and COP9) participate in nucleotide excision repair carried out by the six-factor human excision nuclease.     

The role of DNA polymerase iota in UV mutational spectra

UVB (280-320 nm) and UVC (200-280 nm) irradiation generate predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. CPDs are thought to be responsible for most of the UV-induced mutations. Thymine-thymine CPDs, and probably also CPDs containing cytosine, are replicated in vivo in a largely accurate manner by a DNA polymerase eta (Pol eta) dependent process. Pol eta is a DNA damage-tolerant and error-prone DNA polymerase encoded by the POLH (XPV) gene in humans. Another member of the Y family of error-prone DNA polymerases is POLI encoding DNA polymerase iota (Pol iota). In order to clarify the specific role of Pol iota in UV mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector which replicates in mammalian cells, similar as we have previously done for Pol eta. Synthetic RNA duplexes were used to efficiently inhibit Pol iota expression in 293 T cells. The supF shuttle vector was irradiated with 254 nm UVC and replicated in 293 T cells in presence of anti-Pol iota siRNA. Surprisingly, there was a consistent reduction of recovered plasmid from cells with Pol iota knockdown and this was independent of UV irradiation of the plasmid. The supF mutant frequency was unchanged in the siRNA knockdown cells relative to control cells confirming that Pol iota does not play an important role in UV mutagenesis. UV-induced supF mutants were sequenced from siRNA-treated cells and controls. Neither the type of mutations nor their distribution along the supF gene were significantly different between controls and siRNA knockdown cells and were predominantly C to T and CC to TT transitions at dipyrimidine sites. These results show that Pol iota has no significant role in UV lesion bypass and mutagenesis in vivo and provides some initial data suggesting that this polymerase may be involved in replication of extrachromosomal DNA.     

Impaired translesion synthesis in xeroderma pigmentosum variant extracts

Xeroderma pigmentosum variant (XPV) cells are characterized by a cellular defect in the ability to synthesize intact daughter DNA strands on damaged templates. Molecular mechanisms that facilitate replication fork progression on damaged DNA in normal cells are not well defined. In this study, we used single-stranded plasmid molecules containing a single N-2-acetylaminofluorene (AAF) adduct to analyze translesion synthesis (TLS) catalyzed by extracts of either normal or XPV primary skin fibroblasts. In one of the substrates, the single AAF adduct was located at the 3' end of a run of three guanines that was previously shown to induce deletion of one G by a slippage mechanism. Primer extension reactions performed by normal cellular extracts from four different individuals produced the same distinct pattern of TLS, with over 80% of the products resulting from the elongation of a slipped intermediate and the remaining 20% resulting from a nonslipped intermediate. In contrast, with cellular extracts from five different XPV patients, the TLS reaction was strongly reduced, yielding only low amounts of TLS via the nonslipped intermediate. With our second substrate, in which the AAF adduct was located at the first G in the run, thus preventing slippage from occurring, we confirmed that normal extracts were able to perform TLS 10-fold more efficiently than XPV extracts. These data demonstrate unequivocally that the defect in XPV cells resides in translesion synthesis independently of the slippage process.     

A novel XPC pathogenic variant detected in archival material from a patient diagnosed with Xeroderma Pigmentosum: a case report and review of the genetic variants reported in XPC

The disease Xeroderma Pigmentosum (XP) is genetically heterogeneous and defined by pathogenic variants (formerly termed mutations) in any of eight different genes. Pathogenic variants in the XPC gene are the most commonly observed in US patients. Moreover, pathogenic variants in just four of the genes, XPA, XPC, XPD/ERCC2 and XPV/POLH account for 91% of all XP cases worldwide. In the current study, we describe the clinical, histopathologic, molecular genetic, and pathophysiological features of a 19-year-old female patient clinically diagnosed with XP as an infant. Analysis of archival material reveals a novel variation of a 13 base pair deletion in XPC exon 14 and a previously reported A>C missense pathogenic variant in the proximal splice site for XPC exon 6. Both variations induce frameshifts most likely leading to a truncated XPC protein product. Quantitative RT-PCR also revealed reduced mRNA levels in the archived specimen. Analysis of the XPA, XPD/ERCC2 and XPV/POLH genes in the current specimen failed to reveal pathologic variants. All previously reported pathogenic variants, polymorphisms and known amino acid changes for the XPC gene are compiled and described in the current nomenclature. Given the relative ease of screening for genetic variation and the potential role for such variation in human disease, a proposal for screening appropriate archival materials for alterations in the four most prevalent XP genes is presented.     

FF483–484 motif of human Polη mediates its interaction with the POLD2 subunit of Polδ and contributes to DNA damage tolerance

Switching between replicative and translesion synthesis (TLS) DNA polymerases are crucial events for the completion of genomic DNA synthesis when the replication machinery encounters lesions in the DNA template. In eukaryotes, the translesional DNA polymerase η (Polη) plays a central role for accurate bypass of cyclobutane pyrimidine dimers, the predominant DNA lesions induced by ultraviolet irradiation. Polη deficiency is responsible for a variant form of the Xeroderma pigmentosum (XPV) syndrome, characterized by a predisposition to skin cancer. Here, we show that the FF_483–484 amino acids in the human Polη (designated F1 motif) are necessary for the interaction of this TLS polymerase with POLD2, the B subunit of the replicative DNA polymerase δ, both in vitro and in vivo. Mutating this motif impairs Polη function in the bypass of both an N-2-acetylaminofluorene adduct and a TT-CPD lesion in cellular extracts. By complementing XPV cells with different forms of Polη, we show that the F1 motif contributes to the progression of DNA synthesis and to the cell survival after UV irradiation. We propose that the integrity of the F1 motif of Polη, necessary for the Polη/POLD2 interaction, is required for the establishment of an efficient TLS complex.     

DNA damage induced Pol η recruitment takes place independently of the cell cycle phase

When DNA is damaged in cells progressing through S phase, replication blockage can be avoided by TLS (Translesion DNA synthesis). This is an auxiliary replication mechanism that relies on the function of specialized polymerases that accomplish DNA damage bypass. Intriguingly, recent evidence has linked TLS polymerases to processes that can also take place outside S phase such as nucleotide excision repair (NER). Here we show that Pol η is recruited to UV-induced DNA lesions in cells outside S phase including cells permanently arrested in G1. This observation was confirmed by different strategies including global UV irradiation, local UV irradiation and local multi-photon laser irradiation of single nuclei in living cells. The potential connection between Pol η recruitment to DNA lesions outside S phase and NER was further evaluated. Interestingly, the recruitment of Pol η to damage sites outside S phase did not depend on active NER, as UV-induced focus formation occurred normally in XPA, XPG and XPF deficient fibroblasts. Our data reveals that the re-localization of the TLS polymerase Pol η to photo-lesions might be temporally and mechanistically uncoupled from replicative DNA synthesis and from DNA damage processing.     

Functional lentiviral vectors for xeroderma pigmentosum gene therapy

Xeroderma pigmentosum (XP) is a genetic disease characterized by an autosomal-transmitted genodermatosis involving impaired DNA repair activity, where XP patients present severe sensitivity to sunlight (UVB radiation) and are highly victimized by skin cancer. Complementing XP genes by gene therapy is one potential strategy for helping XP patients. However, current viral-based protocols still lack long-term and stable expression, due to limited post-mitotic infection and gene silencing (in the case of retroviruses) or transient expression and activation of immune response (in the case of adenoviruses). Here we demonstrate that the use of third-generation lentiviral vectors can overcome some of these limitations, rescuing the aberrant phenotype in different categories of the disease (XPA, XPC and XPD). Our results show that lentiviruses are efficient tools to transduce XP fibroblasts and correct repair-defective cellular phenotypes by recovering proper gene expression, normal UV survival and unscheduled DNA synthesis after UV radiation. We propose lentiviral vectors as an attractive alternative for gene therapy protocols focusing on DNA repair genetic diseases.     

Modeling xeroderma pigmentosum associated neurological pathologies with patients-derived iPSCs

Xeroderma pigmentosum (XP) is a group of genetic disorders caused by mutations of XP-associated genes, resulting in impairment of DNA repair. XP patients frequently exhibit neurological degeneration, but the underlying mechanism is unknown, in part due to lack of proper disease models. Here, we generated patientspecific induced pluripotent stem cells (iPSCs) harboring mutations in five different XP genes including XPA, XPB, XPC, XPG, and XPV. These iPSCs were further differentiated to neural cells, and their susceptibility to DNA damage stress was investigated. Mutation of XPA in either neural stem cells (NSCs) or neurons resulted in severe DNA damage repair defects, and these neural cells with mutant XPA were hyper-sensitive to DNA damage-induced apoptosis. Thus, XP-mutant neural cells represent valuable tools to clarify the molecular mechanisms of neurological abnormalities in the XP patients.     

Replication of damaged DNA by translesion synthesis in human cells

Most types of DNA damage block the passage of the replication machinery. In order to bypass these blocks, cells employ special translesion synthesis (TLS) DNA polymerases, which have lower stringency than replicative polymerases. DNA polymerase eta is the major polymerase responsible for bypassing UV lesions in DNA and its absence results in the variant form of the genetic disorder, xeroderma pigmentosum. Other TLS polymerases have specificities for different types of damage, but their precise roles inside the cell have not yet been established. These polymerases are located in replication factories during DNA replication and the polymerase sliding clamp PCNA plays an important role in mediating switching between different polymerases.